Using currently available immunosuppressive approaches, heart transplant recipients experience high incidence of chronic rejection, renal insufficiency, infection, and malignancy. If tolerance could be achieved (permanent acceptance without chronic immunosuppression), each of these important causes of morbidity and mortality could be alleviated. This RFA acknowledges that inducing tolerance to a heart allograft appears to require different approaches than may apply for other organs. This proposal will explore new approaches to modulate an active immune response to donor and heart-specific antigens, based on inhibition of the CD40/CD40L and CD28/B7 costimulation pathways, as an approach to promote peripheral tolerance to a cardiac allograft. In a cynomolgus monkey heart transplant model we find that intensive early blockade of CD 154 or CsA treatment reliably attenuates acute rejection of primate cardiac allografts. However even high-dose ongoing anti-CD154 monotherapy does not prevent cardiac allograft vasculopathy (CAV), which is reliably preceded by production of antidonor alloantibodies (AlloAb), and by increased intracardiac expression of inducible costimulator (ICOS), a costimulation pathway constituent that is expressed following CD28 ligation. Based on these observations, we hypothesize that AlloAb induction and CAV are driven primarily by costimulation-dependent adaptive (acquired) immunity that, in the context of CD154 blockade, is driven by CD28 and ICOS. We predict that more effective control of pathogenic costimulation pathway activation will prevent AlloAb and CAV. This prediction will be tested in Aim 1 using a non-activating scFv anti-CD28 molecule that selectively blocks CD28 signaling, but allows tolerogenic interaction between B7 and CTLA4. This new reagent will be evaluated in the context of three approaches that show promise to induce tolerance in rodents: CsA;CD 154 blockade;and intensive induction T-cell depletion. Aim 2 will extend our prior work with CD 154 blockade, additionally targeting three pathways, CCR5, CD20, and ICOS, that our prior work (and that of others) identifies as integral to alloantibody elaboration in primates. By monitoring immunity to donor antigens and a heart-specific antigen, cardiac myosin, in the context of these two Aims, the experiments proposed will extend our understanding of the role of costimulation in pathogenic and tolerogenic immunity to a heart allograft in a preclinical NHP model. Availability of key reagents is assured through a subcontract.